ProjectSummary?Project2 Therapyresistanceinmelanomaisamajorhurdletoimprovedsurvival.Thisprojectwilldetermineiftherapy resistance can be reversed by targeting the lysosomal enzyme protein palmitoyl thioesterase 1 (PPT1). Autophagy is a lysosome-dependent pathway that promotes tumor growth and resistance to therapy in melanoma.Autophagyinhibitionwithchloroquine(CQ)derivativesaugmentstheefficacyofmanyanticancer therapies, but has limited activity as a single agent. Clinical trials involving HCQ in melanoma show promising activity but concerns have been raised about the potency of HCQ, and its poorly understood mechanism of action. We have prepared dimeric antimalarial compounds that are 10-1000 fold more potent in vitro and in vivo than CQ or HCQ. Dimeric quinacrines (DQs) (Rebecca Cancer Discovery 2017) and dimeric chloroquines (DCs) (Rebecca Cancer Discovery in revision) look especially promising as both tool compounds and potential clinical drugs. In the current cycle, we have found that extending the linker length of these dimeric compounds increases lysosomal localization and anti-melanoma activity. These DQs and DCs with longer linkers as well as CQ were used to pull a new lysosomal target, PPT1, which is overexpressed in cancer, especially in metastatic lesions. Efforts to target the lysosome and autophagy in cancer cells have focused on the effects within cancer cells but recent literature suggests targeting this pathway in immunosuppressive cells within the TME also contributes to antitumor activity. We will leverage innovative collaborations with Projects1, 3 and4 andheavy support from the P01 cores to understand the effects of PPT1 inhibition in both tumor cells, the interaction between tumor cells and fibroblasts, tumor associatedmacrophagesandTcells.Theproposalisbasedonextensivenewpreliminarydatainresponse to the reviewers? critiques from the September 2017 submission. We will test the hypothesis that targeting PPT1 intumor cellsand macrophages overcomes therapy resistance in melanomathrough completion of3 aims: Aim 1 will develop innovative new compounds by introducing heteroatom substitutions into the linker, and developing the first ever dimeric ferroquine derivatives that could have better penetration in the acidic TME. We willalsodevelopanovel assay forPPT1 that is compatible with live cells and animal studies. Aim 2 will leverage collaborations within the P01 to study the role of PPT1 in blocking lipid trafficking from aged fibroblasts to melanoma cells and reversing resistance to targeted therapy. Aim 3 will study the effects of PPT1 inhibition on tumor cell interactions with T cells and macrophages in 3D culture, immunocompetent mouse models, and a new conditional KO model of Ppt1, with the goal of reversing resistance to immunotherapy. The impact of these studies will be to unravel a deeper mechanistic understanding of the consequencesoflysosomalinhibitionwithintheTME,whichwillsupportclinicaldevelopmentoftheseagents inthefuture.Knowledgegainedwillultimatelyleadtoimprovedpatientoutcomes.